CN107110840A - Determined for the SRM of chemotherapy target - Google Patents

Determined for the SRM of chemotherapy target Download PDF

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Publication number
CN107110840A
CN107110840A CN201580045634.1A CN201580045634A CN107110840A CN 107110840 A CN107110840 A CN 107110840A CN 201580045634 A CN201580045634 A CN 201580045634A CN 107110840 A CN107110840 A CN 107110840A
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seq
protein
peptide
rrm1
folr1
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Inventor
大卫·B·克里茨曼
托德·哈姆布拉夫
史诺·塞帕洛姆比尔
辽卫礼
安恩京
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Expression Pathology Inc
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Expression Pathology Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Abstract

The quantitative analysis carried out by Selective reaction monitoring (SRM) mass spectrum/multiple-reaction monitoring (MRM) mass spectrum to the protein as chemotherapy target, the quantitative analysis is carried out by the disclosed specific modified forms from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein of measurement.If the modified forms of peptide include phosphorylated tyrosine, threonine, serine and/or other amino acid residues in its amino acid sequence, these peptides can be quantified.Biological sample fixed in formalin is used for the analysis.

Description

Determined for the SRM of chemotherapy target
The priority for the provisional application 62/019,830 submitted this application claims on July 1st, 2014, its content is by quoting Include in full herein.
Introduction
People kill the cell that is growing and dividing and the therapeutic agent of function is controlled in many ways using a collection of Treat cancer.Common chemotherapeutics set has been used alone or in combination many decades, and the common reagent set turns into and faced Tradition and conventional anti-cancer therapies in bed oncology Practice.These traditional chemotherapeutics all are quickly divided by killing Cell plays a role, and quick division is one of key property of most of cancer cells.This kind of reagent includes coup injury DNA's Alkylating agent；Prevent the taxane of micro-pipe formation；Disturb the antimetabolite of DNA and rna replicon；Disturb enzyme involved by DNA replication dna Anthracene nucleus medicament (antitumor antibiotics)；Prevent the topoisomerase enzyme inhibitor of DNA replication dna；Prevent the breeding of enzyme cellulation required The alkaloid derived from natural products and other compounds of protein；DNA crosslinkings are caused to be closed to suppress DNA replication dna and/or DNA Into the medicine based on platinum.These chemotherapeutics classifications are to suppress the basic function originality exploitation of growth of cancer cells based on it, And its targeting pattern is known little about it.Because they are not that the protein known to selectively targeted and direct suppression is developed , these reagents are not considered " targeting " cancer therapeutic agent in book of time.However, since its exploitation, these reagent classifications In the biochemical function of each be well known and each of which be found for specific protein, enzyme or nucleic acid have There is " targeting " effect." target " of these chemotherapeutics is also well known and is therefore based on whether there is these in a certain given cancer Specific " target " selects the maximally effective chemotherapeutics for the cancer.The embodiment describes the life in test patient source It whether there is the ad hoc approach of these chemotherapy biomarkers in thing sample.
When one or more specific target protein matter provide any or a variety of chemotherapeutics applied to treating cancer to suppress During the index of cancer growth, the targeted approach of cancer therapy is best.This embodiment offers for one or more Peptide and peptide sequence that SRM/MRM is determined, these are determined for quantitatively determining the life directly in the patient source from cancer patient The chemotherapy Protein Index expressed, be overexpressed or do not expressed in thing sample, is determined for the improved treatment for cancer therapy.
The content of the invention
There is provided the particular peptide of the subsequence from following protein：ENT1、ERCC1、FOLR1、RRM1、TUBB3、 TOPO1 and TOPO2A.ENT1 is also referred to as balanced type nucleoside transporter 1 and herein referred to as ENT1.ERCC1 is also referred to as DNA Excision repair protein white matter ERCC-1 and herein referred to as ERCC1.FOLR1 is also referred to as folacin receptor α and is herein referred to as FOLR1.RRM1 is also referred to as ribonucleoside diphosphate reductase large subunit and herein referred to as RRM1.TUBB3 is also referred to as micro-pipe Albumen β -3 chain proteins and herein referred to as TUBB3.TOPO1 is also referred to as DNA topoisomerases 1 and is herein referred to as TOPO1.TOPO2A is also referred to as the α of DNA topoisomerases 2 and herein referred to as TOPO2A.
The peptide sequence and fragment/transition ion of the various peptides from protein can be specifically for based on mass spectrographic selectivity In reaction monitoring (SRM), it is also referred to as multiple-reaction monitoring (MRM) measure, and referred to hereinafter as SRM/MRM.Describing is used for The quantitative SRM/MRM analyses of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and TOPO2A protein and its isotype The purposes of peptide.
Can be used it is one or more, two or more, three or more, four kinds or more kinds, or five kinds or six kinds SRM/MRM determines to measure one kind from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Or the relative or absolute quantitation level of a variety of specific peptides, and thus provide by mass-spectrometer measurement obtained from biological sample to Determine the method for the total amount of each those protein in protein prepared product.It is all or part of available from those protein Peptide also can simultaneously be analyzed or can analyzed in any combination that single SRM/MRM is determined in single SRM/MRM measure.Each peptide is carried The potential method obtained from each corresponding total protein in the given protein prepared product of biological sample by mass-spectrometer measurement is supplied.
More specifically, SRM/MRM of the present invention determine can direct measurement by the cancer trouble fixed from such as formalin Complex proteins cracking prepared by the cell that the patient tissue samples (for example, tumour and biopsy slice of excision) of person's tissue are obtained Those peptides in thing sample.The method for the tissue preparation protein example fixed by formalin is described in U.S. Patent No. 7, In 473, No. 532, its content is incorporated herein by reference in their entirety.The method of patent description, which can be used, is obtained from expression pathology public affairs The Liquid Tissue reagents and scheme for taking charge of (Expression Pathology Inc.) (Rockville, MD) are convenient Ground is carried out.
Carrying out formaldehyde/formalin to the tissue that operation is removed at cancer patient to fix has been connect during pathology are put into practice The conventional method received.As a result, the tissue that formaldehyde/formalin fixes FFPE is most widely available from those patients Organizational form.Formaldehyde/formalin is fixed usually using formalin, and it is herein referred to as formalin." 100% " Formalin is made up of saturated solution (about 40 volume % formalin or 37 weight %) of the formaldehyde in water, and it contains a small amount of use The stabilizer with extent of polymerization is aoxidized in limitation, usually methanol.The most common way of preservation tissue be will entirely be organized in it is logical Soak within (8 hours to 48 hours) for a long time frequently referred in the formalin of 10% neutral buffered formalin, then in room Warm It is lower by fixed whole organization embedding in paraffin so as to long term storage.Therefore, the cancerous tissue that analysis formalin is fixed Molecular analysis methods will be for analyzing the method for most being received and largely being utilized of cancer patient's tissue.
The result that SRM/MRM is determined can be used for association from the patient or tested of wherein collection and preservation tissue (biological sample) The accurate and accurate gauge water of any or all these protein in the specific tissue sample (such as cancer tissue sample) of person It is flat, it can additionally associate potential isotype accurate of these protein and accurately quantify level.This is not only provided on cancer The diagnostic message of disease, and allow that doctor or other medical professionals determine the appropriate therapy for patient or object.There is provided On the diagnosis of protein expression level in illing tissue or another patient/subject sample and this survey for the treatment of important information It is fixed to be referred to as with diagnostic assay.For example, this measure may be configured to diagnose stage, degree or the histologic characteristics of cancer and true It is fixed to grow most effectively tissue cancer cell so that the therapeutic agent of patient or object most probable response.More specifically, to coming From one kind of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein in the cancer cell of patient or It is a variety of, two or more, it is three or more, four kinds or more plant, five kinds or more kinds are detected and/or can quantitatively be carried For some protein, this proteinoid can show should follow which kind of or which plant therapeutic scheme.
Detailed description of the invention
Measure of the present invention, which quantifies or measured to come from, includes ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 And/or the relative or abswolute level of the specific unmodified peptide of the protein of TOPO2A protein and also measurable from those The relative or abswolute level of the peptide of the specific modification of protein.The example of modification is included in phosphorylation amino present on these peptides Sour residue and glycosylated amino acid residues.The relative quantification level of protein and protein isoforms can pass through SRM/MRM methods It is determined that, such as by comparing SRM/MRM characteristic peak areas (signature peak area) (for example, characteristic peak area or integration Fragment ions intensity) determine.Can be at different samples (for example, control sample and the sample prepared by patient or object tissue) The middle relative level for determining single ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide.Or, each In the case that kind of peptide has the specific SRM/MRM characteristic peaks of their own, can compare a variety of one or more ENT1, ERCC1, Multiple SRM/MRM characteristic peak areas of FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A feature peptide.Pass through comparison peak face Product, can determine in a kind of biological sample or the ENT1 in one or more extra or different biological samples, ERCC1, The relative level of FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein and potential protein isotype content.With this Mode, can be under same experimental conditions across multiple (for example, two, three, four, five or more) Determination of Biological Samples The relative amounts of specific one or more peptides from those protein and thereby determine ENT1, ERCC1, FOLR1, RRM1, The relative amount of TUBB3, TOPO1 and/or TOPO2A protein and its potential isotype.In addition, determining to come from simple sample The one or more of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein give the relatively fixed of peptide The method of amount can compare the characteristic peak area and the same protein system come biological sample of the peptide by SRM/MRM methods Characteristic peak area of other from different one or more protein with different one or more peptides in standby thing.Use This kind of method, can determine the specific peptide from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Content, and then determine the content of each corresponding protein and its potential isotype, the mode of the measure is a kind of protein phase For another protein in same sample or different samples.Because the relative quantification of single one or more peptides can be relative to In sample or the content of another or a variety of peptides of sample room is measured, therefore the relative of existing peptide can be determined contain Amount (such as by determine one kind relative to alternative peak area), this absolute weight with protein in biological sample：Volume Or weight：Weight content is unrelated.Therefore can be used the ENT1 come in the protein prepared product of biological sample, ERCC1, FOLR1, The content of RRM1, TUBB3, TOPO1 and/or TOPO2A peptide come determine among several samples or between those protein contain Amount.The relative quantification data of single feature peak area are generally normalized to institute's analysing protein in unit sample between different samples Content (for example, carrying out normalized sample using the total protein concentration and the volume analyzed of sample).It may span across from single Simultaneous ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein and multiple albumen in sample Many peptides of matter and/or relative quantification is carried out across many samples, so as to further appreciate that relative protein content, i.e., relatively In a kind of peptide/protein of other peptide/protein.
The absolute quantitation level of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein passes through For example SRM/MRM methods are determined, by the ENT1 in this method in the future a kind of comfortable biological sample, ERCC1, FOLR1, RRM1, The SRM/MRM characteristic peak areas of the single peptide of TUBB3, TOPO1 and/or TOPO2A protein and one or more internal standard compounds are Know that the SRM/MRM characteristic peak areas of content compare, these internal standard compounds with known content " incorporation " sample (for example, isotope The standard items of mark).In one embodiment, the internal standard compound be the identical ENT1 of synthesized form, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide, it contains one or many of useful one or more heavy labels Individual amino acid residue.Synthesize the internal standard compound of this kind of isotope marks so that predictable and consistent by being produced during mass spectral analysis SRM/MRM characteristic peaks, its with natural ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide characteristic peak not It is same and completely different and can be used as comparison peak.Therefore, when internal standard compound with known content incorporation come biological sample protein or During in peptide prepared product and by mass spectral analysis, by the SRM/MRM characteristic peak areas of native peptides and the SRM/MRM characteristic peaks of internal standard peptide Area compares.The numeric ratio is compared with allowing to calculate the absolute of native peptides present in the urporotein preparation of biological sample Molar concentration and/or absolute weight, can therefrom determine the concentration or weight of corresponding protein.The absolute quantitation data of fragment peptide are led to Often shown according to the content for the protein analyzed in every kind of sample.Absolute quantitation may span across many peptides progress, and (it allows fixed Simultaneous multiple proteins (for example, two kinds, three kinds, four kinds, five kinds etc.) in single sample are determined in measurement) and/or cross over many Plant sample to carry out, so as to understand the absolute protein content in single biological sample and/or in the group of whole single sample.One In individual embodiment, Gyai etc. can be used in United States Patent (USP) 7, the poly saccharide peptide standard product described in 501,286 carries out determining for protein Amount.
Herein, term is quantitative, quantify, measurement or measurement refer to the relative or abswolute level for determining analyte, the analyte It is such as protein, polypeptide, peptide, standard items (such as internal standard compound).Of the present invention determine can be used for using such as patient Auxiliary determines carcinoma stage during tissue (tissue that such as formalin is fixed) of source or object origin.SRM/ of the present invention MRM, which is determined, can also be used to aid in determining which kind of therapeutic agent will be most advantageously used for treating the patient or object.
, can be to via the part or all of tumour of removal to detect the level with cancer-associated proteins matter of the present invention Operation or via the doubtful cancer removed from patient or object for the biopsy method that determines the presence or absence of suspected disease and carry out Property tissue or cancerous tissue are analyzed.The sample of these tissues is analyzed to determine whether there is suspected disease.Analyze this It whether there is ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 in sample of the sample organized a bit to determine patient or object And/or TOPO2A protein and these protein that there is which kind of form.Furthermore, it may be determined that a kind of or many in these protein The expression of kind simultaneously compares it with " normal " or reference levels found in health tissues.Found in health tissues Protein normal or reference levels can from for example without cancer one or more individual linked groups.Or, It can be obtained by analyzing linked groups' (for example, part of same organs) of non-affected by cancer with the individual of cancer Normal or reference levels.
The level or content of protein or peptide may be defined as determining the amount (table of the protein determined or peptide by SRM/MRM Be shown as mole, quality or weight).The level or content can be normalized to protein in the lysate of analysis or another composition Aggregate level or content (for example, being expressed as micromole/micrograms of protein or microgram/micrograms of protein) are even normalized to list DNA content (for example, micromole or microgram/micrograms of DNA) on the basis of the weight of position.In addition, the level or content of protein or peptide Can be determined with volume basis, be for example expressed as micromole or nanogram/microlitre.The protein or peptide that determine are determined by SRM/MRM Level or content can also be normalized to the number of analyzed cell.Be related to ENT1, ERCC1, FOLR1, RRM1, TUBB3, The information of the isotype of TOPO1 and/or TOPO2A protein and these protein can be used for auxiliary determine cancer stage or Grade, method is association or compares ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein and its same The level observed in the level and normal structure of the type of kind or fragment peptide.Once it is determined that the histology stage of cancer and/or grade And/or ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein expression profile, then can be by the information With being matched through developing with the list of (chemistry and biology) therapeutic agent of specific treatment cancerous tissue, the feature of the cancerous tissue Be for example determined one or more protein (such as ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A unconventionality expression).Match ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 from particular individual and/or The information of TOPO2A protein determinations with it is selectively targeted expression ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The therapeutic agent list of the cell/tissue of TOPO2A protein represents the personalized medicine method for the treatment of cancer.It is of the present invention Assay method use the protein from patient or object autologous tissue analysis as diagnosis and treatment decision source, from And form the basis of personalized medicine method.
Peptide is generated
In principle, by the known proteolysis process of any specificity prepare from ENT1, ERCC1, The peptide of any prediction of FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein can be employed as alternative report to survey Determine the abundance of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein.In one embodiment, Use one or more protease known to specificity (such as one or more trypsase and/or intracellular protein enzyme Lys-C) Sample digestion.One or more peptides that proteolytic treatment is obtained can be in the suitable survey such as determined based on mass spectrographic SRM/MRM Be used as alternative report in fixed determine one or more ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The abundance of TOPO2A protein.Similarly, it is possible to use it is known in ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and The peptide sequence for containing any prediction of an amino acid residue at the site being modified in TOPO2A protein comes in determination sample The degree of modification of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and TOPO2A protein.
ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A fragment peptide can be produced by a variety of methods It is raw, including the use of the Liquid Tissue provided in United States Patent (USP) 7,473,532TMScheme.Liquid TissueTMScheme The paraffin that can be fixed with reagent by carrying out proteolytic digestion to the protein in tissue/biological sample by formalin Peptide sample of investing tissue's generation suitable for mass spectral analysis.In Liquid TissueTMIn scheme, tissue/Biological preparation is existed A period of time of extension is kept (for example, about 80 DEG C to about 100 DEG C, to last about 10 minutes to about 4 in buffer solution and elevated temperature Hour time) to reverse or discharge protein cross.The buffer solution used is neutral buffered liquid (for example, based on Tris's Buffer solution or the buffer solution containing detergent) and preferably do not disturb the buffer of mass spectral analysis.Then, by tissue/biological sample At one or more protease with including but not limited to trypsase, chymotrypsin, pepsin and intracellular protein enzyme Lys-C Reason is enough to destroy the tissue and eucaryotic cell structure of the biological sample and makes the sample liquefied time (for example, at about 37 DEG C extremely Last time of about 30 minutes to about 24 hours at a temperature of about 65 DEG C).The result of heating and proteolysis is soluble for liquid Dilutable biomolecule lysate.In one group of embodiment, selected from trypsase, chymotrypsin, pepsin and intracellular Protease Lys-C two or more protease are used for the proteolytic treatment of biological sample.
Peptide is separated and determined
Once prepare lysate, so that it may to the peptide in sample using a variety of skills for promoting its analysis and measuring (quantitative) Art.When being analyzed by mass spectrum, one or more chromatographic process can be used to promote analysis.
In one embodiment, peptide is separated by liquid chromatogram (LC) before being analyzed by mass spectrometer.Example Such as, PicoFrit (100 μm of ID/10 μm of tip ID, fresh target company (New Objective)) post can be used to exist (match is silent to fly by nanoAcquityLC systems (Waters (Waters), Massachusetts Penelope Milford) or EASY-nLC II Your scientific ＆ technical corporation of generation (Thermo Scientific), San Diego, CA) on isolated peptides, the post uses Jupiter ProteoC12,4 μm of resins (Féraud door company (Phenomenex), California Tuo Lunsi) are from filling to 12cm Bed length.Can be in 12 minutes with the chromatogram gradient (containing 0.1% formic acid) of 1% to 50% acetonitrile under 800nL/ minutes flow velocitys Elute peptide.Once being separated by liquid chromatogram, then the peptide of elution can be imported into mass spectrum is analyzed.In one embodiment, Mass spectrum is equipped with nano-spray source (nanospray source).
In another embodiment, by affine technolog isolated peptides, such as based on immune purifying (such as affine in immunity color Spectrum), the chromatogram on ion selectivity medium, or separated when peptide is modified by using appropriate medium, the medium is example Such as it is used for the agglutinin for the peptide that separating carbohydrates are modified.In another embodiment, SISCAPA methods have been used, its The immune separation of peptide is carried out before mass spectral analysis.The SISCAPA technologies are described in such as U.S. Patent number 7,632,686.Another In one embodiment, agglutinin affine method (such as affinity purification and/or color can be used before being analyzed by mass spectrum Spectrum) carry out the isolated peptides from lysate.The method (including method based on agglutinin) of isolated peptides group is described in such as Geng J.Chromatography B, 752：293-306(2001).Immune affinity chromatographic technology, agglutinin affine technolog and other shapes The affine separation of formula and/or chromatogram (separation such as anti-phase, based on size, ion exchange) can suitably be applied in combination to promote Peptide is analyzed by mass spectrum.
Surprisingly it was found that from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A albumen Many potential peptide sequences of matter are not appropriate for using or based on mass spectrographic SRM/ in determining based on mass spectrographic SRM/MRM MRM is failure when being used in determining, and its reason is failed to understand.Specifically, find being fixed from formalin, FFPE The Liquid Tissue of tissueTMIn lysate can not effective detection or can not detect at all it is many from ENT1, ERCC1, The tryptic peptide of FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein.Due to most suitable MRM/ can not possibly be predicted The peptide that SRM is determined, it is therefore necessary to by testing in actual Liquid TissueTMIdentify modifying and unmodified in lysate Peptide with develop be directed to ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein it is reliable and accurate SRM/MRM determine.It is not intended to by any theoretical constraint, but thinks that some peptides for example may be difficult to by Mass Spectrometer Method, because They will not ionize or generate well the fragments different from other oroteins, and peptide be able to not may also be being separated (for example, liquid phase color Spectrum) in parse well, or be attached on glass or plastic ware.Therefore, can be in the tissue sample fixed by formalin The Liquid Tissue of preparationTMDetected in lysate from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or Those peptides (for example, peptide in table 1 and 2) of TOPO2A protein are in ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 And/or TOPO2A protein s RM/MRM determine in can pin be used for SRM/MRM measure peptide.In one embodiment, exist Used when separating ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein simultaneously in single sample Protease will be trypsase.
ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 for being found in multiple embodiments of the present invention and/ Or TOPO2A peptides (for example, table 1 and/or table 2) pass through the complicated Liquid Tissue of Trypsin InducedTMInstitute in lysate Have protein and from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein, the lysate by It is prepared by the cell obtained in the cancerous tissue fixed from formalin.Unless otherwise noted, otherwise in all cases, the albumen Enzyme is all trypsase.Liquid TissueTMLysate then by mass spectral analysis with determine by Mass Spectrometer Method and analyze From those peptides of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein.For mass spectral analysis The identification of specific preferred peptide subgroup be based on：1) in Liquid TissueTMWhat is ionized in the mass spectral analysis of lysate comes from egg The experiment of one or more peptides of white matter is determined, and 2) peptide is preparing Liquid TissueTMThe scheme and reality used in lysate The ability existed under the conditions of testing.The scope of latter property is not only the amino acid sequence of peptide, and is the modification in peptide The ability that amino acid residue is existed during sample preparation in the form of modification.
The protein cleavage thing of the cell directly obtained from the fixed tissue of formalin (formaldehyde) uses Liquid TissueTMPrepared by reagent and scheme, Liquid TissueTMReagent and scheme requirement collect cell via Tissue microdissection Into sample cell, then in Liquid TissueTMA period of time of extension is heated in buffer solution to cell.Once negatively shadow The crosslinking induced to formalin is rung, then is disappeared using protease (such as including but is not limited to trypsase) in predictable form Change tissue/cell to complete digestion.Each protein cleavage thing is changed into peptide by using the complete polypeptide of protease digestion Set.Analyze (for example, by ion trap mass spectrometry) each Liquid TissueTMLysate is with to the multiple albumen of overall importance of peptide progress Group learns research, and wherein data are expressed as being reflected by mass spectrum for all cell proteins present in each protein cleavage thing The qualification result of fixed peptide as much as possible.Generally use ion trap mass spectrometry or another shape of profiling of overall importance can be carried out The mass spectrum of formula come identify the peptide as much as possible from single complex proteins/peptide lysate for analysis.Although can be in bag Include and developed on MALDI, ion trap or the mass spectrographic any kind of mass spectrum of triple quadrupole and carry out SRM/MRM measure, but generally recognize It is triple quadrupole mass spectrometer platform for best instrument platform for being determined for SRM/MRM.
Once identify peptide as much as possible in the single MS analyses of single lysate under the applied conditions, then it is whole Manage the list of peptide and use it for the protein that determination is detected in the lysate.For a variety of Liquid TissueTMCracking Thing repeats the process, and very big peptide list is organized into single data set.The data set of the type can be considered as into representative can In the biological sample type of analysis (after protease digestion), particularly in the Liquid Tissue of biological sampleTMLysate In the peptide that detects, and therefore include such as ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Specific protein peptide.
In one embodiment, ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A trypsase Peptide is accredited as the absolute or relative amount that can be used for determining following peptide：ENT1 is (for example, NCBI accession number Q99808, SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO： 7), ERCC1 is (for example, NCBI accession number P07992, SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16), FOLR1 (for example, NCBI accession number P15328, SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20), RRM1 is (for example, NCBI accession number P23921, SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO： 24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37), TOPO1 is (for example, NCBI accession number P11387, SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56、SEQ ID NO：57、SEQ ID NO：58), TOPO2A is (for example, NCBI accession number P11388, SEQ ID NO：59、SEQ ID NO：60、SEQ ID NO： 61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64、SEQ ID NO：65、SEQ ID NO：66、SEQ ID NO：67、SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74、SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78.SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98、SEQ ID NO：99、SEQ ID NO：100、SEQ ID NO：101) and/or TUBB3 (for example, NCBI accession number Q13509, SEQ ID NO：102、SEQ ID NO：103), it is each listed in table 1.Each leisure of those peptides is by good fortune Your Malin fixes, the Liquid Tissue of the tissue preparation of FFPETMPass through Mass Spectrometer Method in lysate.Therefore, in table 1 Each peptide or those peptides any combination (for example, it is one or more, two or more, it is three or more, four kinds or more Kind, five kinds or more plant, six kinds or more plant, seven kinds or more plant, eight kinds or more plant, nine kinds or more kind, or ten kinds or Those peptides in more kinds of tables 1) it is for ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein The candidate that quantitative SRM/MRM is determined, including be directly measured in the patient or object tissue that formalin is fixed.
Table 1
ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A tryptic peptide listed in table 1 include The a variety of Liquid for the tissue fixed from a variety of different formalin of the different human organs including prostate, colon and mammary gland TissueTMThose detected in lysate.Those peptides be each considered as can be used for formalin fix tissue in ENT1, The quantitative SRM/MRM of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein is determined.What these were tested enters one Step data analysis shows, do not observe any particular peptide from any certain organs site preferentially.Therefore, in these peptides Each be considered as being adapted to from any Fu Er from any biological sample or internal any organ site The Liquid Tissue for the tissue that Malin fixesTMLysate carry out ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/ Or the SRM/MRM of TOPO2A protein is determined.
In another embodiment, SRM/MRM, which is determined, uses the respective one or two kinds of peptide (examples of TOPO2A and TOPO1 Such as, the peptide enumerated in table 1).In another embodiment, SRM/MRM, which is determined, uses ENT1, ERCC1, FOLR1, RRM1 And/or the respective one or two kinds of peptides (for example, the peptide enumerated in table 1) of TUBB3.
In other embodiments, two kinds of one of measurement ENT1 and ERCC1 protein or whole are determined simultaneously using SRM/MRM Determine FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein in one kind, two kinds, three kinds or four kinds.In this kind of reality In the one embodiment for applying mode, two kinds of one of measurement FOLR1, RRM1 protein or whole are determined at least by SRM/MRM A kind of peptide or at least two peptides (such as FOLR1, RRM1 peptide enumerated in table 1)；And determine ENT1, ERCC1, TUBB3, TOPO1 And/or any one in TOPO2A, two kinds, three kinds or four kinds of at least one peptide or at least two peptides in table 1 (for example, enumerate Peptide).In another embodiment of this kind of embodiment, by SRM/MRM determine measurement one of ENT and RRM1 protein or Whole two kinds at least one or at least two peptides (such as the peptide enumerated in table 1)；And determine ERCC1, FOLR1, TUBB3, Any at least one or at least two peptides (for example, the peptide enumerated in table 1) in TOPO1 and/or TOPO2A.The present invention is provided The composition of peptide is included, these peptides are through any in isotope marks but other these embodiments provided with the present invention Shown one or more peptides are identical, and its prepared product application is described below, particularly as mass spectrum standard items.
In one embodiment, by determining peptide or those peptides in one or more tables 1 independent of mass spectrographic method Any combination (for example, it is one or more, two or more, it is three or more, four kinds or more plant, five kinds or more Kind, six kinds or more plant, seven kinds or more plant, eight kinds or more plant, nine kinds or more kind), this method includes but is not limited to exempt from Epidemic disease method (such as Western markings or ELISA).In one embodiment, the tissue fixed using formalin is surveyed It is fixed.The information for being related to peptide (definitely or relative) content is obtained anyway, and the information is used equally for any means of the present invention, Including the presence of cancer, the stage/grade/state for determining cancer, offer prognosis in display (diagnosis) patient or object, or determine For patient or the therapeutic agent or therapeutic scheme of object.
In other embodiments, by determining one of ENT1 and ERCC1 protein or whole independent of mass spectrographic method Two kinds, and determine one kind in FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein, two kinds, three kinds or four kinds, should Method includes but is not limited to immunization method (such as Western markings or ELISA).In one embodiment of this kind of embodiment In, one of ENT1 and ERCC1 protein or whole two kinds at least one or at least two peptides are determined (for example, being enumerated in table 1 ENT1 and ERCC1 peptides)；And determine in FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein any one, Two kinds, three kinds or four kinds of at least one or at least two peptides (for example, the peptide enumerated in table 1).In the another of this kind of embodiment In one embodiment, one of FOLR1 and RRM1 protein or all two kinds of at least one or at least two peptide (examples are determined Such as, FOLR1 the and RRM1 peptides enumerated in table 1)；And determine in ENT1, ERCC1, TUBB3, TOPO1 and TOPO2A protein appoint A kind of at least one or at least two peptides (for example, the peptide enumerated in table 1).
When carrying out SRM/MRM measure, one important Consideration is the type of the instrument available for peptide analysis.Although It can be developed on including MALDI, ion trap or the mass spectrometric any kind of mass spectrograph of triple quadrupole and carry out SRM/MRM surveys It is fixed, but it is typically considered triple quadrupole instrument platform currently used for the SRM/MRM best instrument platforms determined.The type Mass spectrum be often considered be best suited for analysis can by from it is intracellular containing all proteins it is tens thousand of to millions of individual single The target peptide of single separation in the very complicated protein cleavage thing of peptide composition.
In order to from the various of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Peptide is most effectively implemented SRM/MRM and determined, it is necessary to utilize the information in addition to peptide sequence in analysis.The extraneous information can use Mass spectrum (for example, triple quadrupole mass spectrum) is and guided to carry out analysis that is correct and concentrating to specific target peptide in guiding, so as to Effectively it is measured.
On target peptide totally and on specific ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A The extraneous information of peptide may include single isotopic mass of each peptide, its preceding body charge state, precursor m/z values, m/z transition ions and each One, two, three, four or more in the ionic type of transition ion.Available for exploitation for ENT1, ERCC1, The additional peptide information that the SRM/MRM of FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein is determined is shown in Table 2, its ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide are planted for 12 (12) enumerated in table 1.It can make It is standby, obtain be directed to the peptide that shows is described in table 2 the extraneous information and apply it to from ENT1, ERCC1, FOLR1, In the analysis of any other peptide of RRM1, TUBB3, TOPO1 and/or TOPO2A protein, including pass through other protease or egg Those peptides of white enzyme combination (such as trypsase and/or Lys C) generation.
Table 2
In some embodiments, it is adaptable to ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A egg The peptide (such as the peptide shown in table 1 and shown in SED ID No.1-103) of the measure of white matter can contain the extra egg inside peptide sequence White hydrolytic sites, it will generate sub- peptide (sub-peptide) when cut.This kind of sub- peptide can be by assessing for required albumen The sequences of the identified peptides of the proteolytic cleavage sites of enzyme is recognized.In one embodiment, tryptic peptide may include Extra inside positioned trypsin cleavage site, it can generate sub- peptide after by trypsase further cut.
In another embodiment, tryptic peptide can the internal site containing protease, the protease includes but not Be limited to trypsase, GluC, AspN, chymotrypsin and/or Lys C, its can cause by trypsase, GluC, AspN, In chymotrypsin and/or Lys C any one, form sub- peptide after two or more cuttings.In another embodiment party In formula, Lys C peptides can the internal site containing other protease, other protease are such as GluC, AspN, chymotrypsin protein Enzyme and/or trypsase, its can cause by any one in GluC, AspN, chymotrypsin and/or trypsase, Sub- peptide is formed after two or more cuttings.It should be understood that this kind of sub- peptide, and peptide specifically shown in SEQ ID No.1-103 In any one or more trypsase, GluC, AspN, chymotrypsin and/or Lys C cutting fragment, be included in and wrap Containing within the scope of the invention.
Listed embodiment of the invention includes composition, and it includes the peptide in one or more tables 1 and 2 and optionally wrapped Containing through isotope marks but other aspects and the peptide identical peptide in one or more tables 1 and 2.In some embodiments, this A little compositions comprising it is one or more, two or more, it is three or more, four kinds or more plant, five kinds or more plant, six Kind or more plant, seven kinds or more plant, eight kinds or more plant, nine kinds or more kind, or all 103 (103) plant table 1 and 2 In peptide.This based composition optionally include peptide, polypeptide or protein, its amino acid sequence include through isotope marks but its With the peptide identical peptide in one or more Tables 1 and 2s in terms of him.Using comprising it is a kind of, two kinds, three kinds, four kinds, five kinds, six Kind or more when planting in table 1 and 2 synthesis through isotope marks of peptide or native peptides, polypeptide or protein, Protease Treatment is released Put through isotope marks but other aspects and the peptide identical peptide in table 1 and 2.
For example, the amino acid of isotope marks can be used to prepare this in the cell lysate or tissue culture of sequencing The biology or biosynthesis peptide of class isotope marks.One or more isotope marks can be used in the peptide of each isotope marks, this A little isotopes are independently selected from 18O, 17O, 34S, 15N, 13C, 2H or its combination.Regardless of whether through isotope marks, comprising next It need not contain from the composition of the peptide of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein There is the peptide (such as complete tryptic peptide group) from the protein.In some embodiments, these compositions are not contained All peptides of combining form from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein, and tool The peptide occurred in all Tables 1 and 2s is not contained for body.Composition comprising peptide can be dried or lyophilized material, liquid The form of (such as aqueous) solution or suspension, array or the marking.
In one embodiment it is directed to specific ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A The extraneous information of peptide include one or more of, two or more, it is or three or more：Single isotope matter of each peptide Amount, its preceding body charge state, precursor m/z values, m/z transition ions, and ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 And/or the ionic type of each transition ion of the Lys C proteins hydrolysis gained peptide of TOPO2A protein.
In another embodiment, be related to specific ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The extraneous information of TOPO2A peptides include one or more of, two or more, it is or three or more：The Dan Tongwei of each peptide Quality amount, its preceding body charge state, precursor m/z values, m/z transition ions, and ENT1, ERCC1, FOLR1, RRM1, TUBB3, The ionic type of each transition ion of peptide obtained by the trypsin proteolysis of TOPO1 and/or TOPO2A protein.
In another embodiment, be related to specific ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The extraneous information of TOPO2A peptides include one or more of, two or more, it is or three or more：The Dan Tongwei of each peptide Quality amount, its preceding body charge state, precursor m/z values, m/z transition ions, and ENT1, ERCC1, FOLR1, RRM1, TUBB3, The ionic species of each transition ion of trypsase and/or Lys C proteins the hydrolysis gained peptide of TOPO1 and/or TOPO2A protein Type.In one embodiment, from each ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Single trypsase and/or Lys C proteins range of hydrolysed peptides and related extra be used for diagnosis and determine (diagnostic determination)。
Embodiment
The embodiment of the present invention is as follows.
1. ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A albumen in one kind measurement biological sample The method of the level of matter, including the use of in Mass Spectrometer Method and/or the quantitative protein digestibility thing prepared by the biological sample one Kind or a variety of modifications and/or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein The content of fragment peptide；And calculate ENT1 modified in the sample or unmodified, ERCC1, FOLR1, RRM1, TUBB3, The level of TOPO1 and/or TOPO2A protein；And the content is relative amount or absolute content.
2. the method for embodiment 1, is additionally included in detection and/or quantitative one or more modifying or unmodified To the protein before the content of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide The step of digest is classified.
3. the method for embodiment 2, wherein the classification step is selected from gel electrophoresis, liquid chromatogram, Capillary Electrophoresis, received Rice reverse phase liquid chromatography, high performance liquid chromatography or reverse high performance liquid chromatography.
4. any one of embodiment 1-3 method, wherein the protein cleavage thing of the biological sample passes through Liquid TissueTMIt is prepared by scheme.
5. any one of embodiment 1-3 method, wherein the protein digestibility thing includes protease digestion thing.
6. the method for embodiment 5, wherein the protein digestibility thing includes trypsase and/or lys C digests.
7. any one of embodiment 1-6 method, wherein the mass spectrum include tandem mass spectrum, it is ion trap mass spectrometry, triple Four-electrode spectrum, MALDI-TOF mass spectrums, MALDI mass spectrums and/or flight time mass spectrum.
8. the method for embodiment 7, wherein the mass spectrum pattern used is Selective reaction monitoring (SRM), multiple-reaction monitoring And/or more options reaction monitoring (mSRM), or its any combination (MRM).
9. any one of embodiment 1-8 method, wherein ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/ Or TOPO2A protein fragments peptide includes SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56.SEQ ID NO：57、SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64、SEQ ID NO：65、SEQ ID NO：66、SEQ ID NO：67、SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74、SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78.SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98、SEQ ID NO：99、SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102 and SEQ ID NO：Amino acid sequence shown in 103.
10. any one of embodiment 1-9 method, the wherein biological sample are blood sample, urine samples, serum sample Product, ascites sample, tired liquid sample, lymph, saliva sample, cell or solid tissue.
11. any one of embodiment 1-10 method, the wherein biological sample are the tissues that formalin is fixed.
12. any one of embodiment 1-11 method, the wherein biological sample are the tissues of FFPE.
13. any one of embodiment 1-12 method, the wherein biological sample are the tissues obtained from tumour.
14. the method for embodiment 13, the wherein tumour are primary tumors.
15. the method for embodiment 13, the wherein tumour are secondary tumorses.
16. any one of embodiment 1-15 method, in addition to quantitative modification and/or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide.
Any one of 17 (a) embodiments 1-16 method, wherein quantitatively ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide include in a kind of relatively biological sample comprising ENT1, ERCC1, FOLR1, RRM1, About the 8 of TUBB3, TOPO1 and/or TOPO2A protein to the amino acid sequence of about 45 each amino acid residues one or more The content of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide from it is different and individually Identical ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide in sample or biological sample Content.
Any one of 17 (b) embodiments 1-16 method, wherein quantitatively ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide include in a kind of relatively biological sample comprising ENT1, ERCC1, FOLR1, RRM1, About the 8 of TUBB3, TOPO1 and/or TOPO2A protein to the amino acid sequence of about 45 amino acid residues one or more The content of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide from it is different and individually Identical ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide in sample or biological sample Content, the fragment peptide is shown in SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO： 11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48、SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56.SEQ ID NO：57、SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64、SEQ ID NO：65、SEQ ID NO：66、SEQ ID NO：67、SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74、SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78.SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98、SEQ ID NO：99、SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102 and SEQ ID NO：103.
18. the method for embodiment 17 (a) or 17 (b), wherein quantitative one or more ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide include by the internal standard peptide with the known content of addition compared come Determine each in ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide in biological sample The content planted, each ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A albumen wherein in the biological sample Matter fragment peptide is compared with the internal standard peptide of the addition with same amino acid sequence.
19. the method for embodiment 18, the wherein internal standard peptide are the peptides of isotope marks.
20. the internal standard peptide of the method for embodiment 19, the wherein isotope marks include selected from 18O, 17O, 34S, 15N, 13C, 2H or its combination one or more weight stable isotope.
21. any one of embodiment 1-20 method, wherein it is a kind of in detection and/or the quantitative protein digestibility thing or A variety of modifications or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide Content represent in the presence of modification and/or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A Protein and the correlation with cancer in patient or object.
22. the method for embodiment 21, in addition to associate it is a kind of in the detection and/or the quantitative protein digestibility thing or A variety of modifications and/or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments The result of the content of peptide or the content of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein With diagnostic phases/grade/state of cancer.
23. the method for embodiment 22, wherein associating a kind of or many in the detection and/or the quantitative protein digestibility thing Plant modification or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide The result and cancer of content or the content of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein The content of peptide of the diagnostic phases/grade/state of disease with detection and/or other quantitative protein or from other protein is with more Double recipe formula is combined, so as to provide the extraneous information of diagnostic phases/grade/state on cancer.
24. any one of embodiment 1-23 method, in addition to treated for patient or Object Selection, the biological sample Product are obtained from the patient or object, and the treatment is based on one or more ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 And/or TOPO2A protein fragments peptides presence, be not present or content or ENT1, ERCC1, FOLR1, RRM1, TUBB3, The content of TOPO1 and/or TOPO2A protein.
25. any one of embodiment 1-24 method, in addition to give to patient or object the treatment of therapeutically effective amount Agent, the biological sample is obtained from the patient or object, wherein the therapeutic agent and/or the content of therapeutic agent given be based on it is a kind of or A variety of modifications or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide Content or ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein content.
26. the method for embodiment 24 and 25, the wherein treatment or the therapeutic agent for expression ENT1, ERCC1, FOLR1, The cancer cell of RRM1, TUBB3, TOPO1 and/or TOPO2A protein.
27. embodiment 1-27 method, the wherein biological sample are the tumor tissues that the formalin through processing is fixed, It is described to handle for using Liquid TissueTMScheme and reagent quantitative one or more are modified or unmodified ENT1, The content of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide.
28. any one of embodiment 1-28 method, wherein one or more modify or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide are the peptides in one or more tables 1.
29. a kind of composition, its comprising it is one or more, two or more, it is three or more, four kinds or more Kind, five kinds or more plant, six kinds or more plant, seven kinds or more plant, eight kinds or more plant, nine kinds or more kind, or ten kinds or Peptide and/or its antibody in more kinds of tables 1.
30. the composition of embodiment 30, its comprising it is one or more, two or more, it is three or more, four kinds Or more plant, five kinds or more plant, six kinds or more plant, seven kinds or more plant, eight kinds or more plant, nine kinds or more kind, or Peptide and/or its antibody in ten kinds or more kind tables 2.
Exemplary SRM/MRM assay methods
Methods described below is used for：1) identification available for ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein based on mass spectrographic SRM/MRM determine from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/ Or the candidate peptide of TOPO2A protein, 2) it is directed to from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A The target peptide of protein develops that single SRM/MRM is determined or a variety of SRM/MRM are determined, and 3) examines quantitative determination applied to cancer The selection of disconnected and/or optimal therapy.
The SRM/MRM candidate segments of 1.ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein The identification of peptide：
A. digesting protein (is potentially included or trypsase may not be included) using one or more protease, so that by The biological sample that formalin is fixed prepares Liquid TissueTMProtein cleavage thing
B. Liquid Tissue are analyzed on ion trap tandem mass spectrometryTMAll proteins fragment in lysate is simultaneously identified All fragment peptides from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein, wherein single Section peptide is modified without any such as phosphorylation or glycosylated peptide
C. Liquid Tissue are analyzed on ion trap tandem mass spectrometryTMAll proteins fragment in lysate is simultaneously identified Come come with such as phosphorylation or glycosylated residues peptide modify ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/ Or all fragment peptides of TOPO2A protein
D. it is measurable by complete total length ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A albumen Matter is by formal by the issuable all peptides of specific digestion method, but for developing the preferred peptide that SRM/MRM is determined Complicated Liquid Tissue prepared by the biological sample that woods is fixedTMIn protein cleavage thing by mass spectrum Direct Identification those
E. the Liquid Tissue for the biological sample fixed in analysis from formalinTMDuring lysate will in patient or Special sex modification (phosphorylation, glycosylation etc.) and the ionization and peptide that therefore can be detected is accredited as in mass spectrum in object tissue For determining the candidate peptide that the peptide of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein is modified
2. the matter of the fragment peptide from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Spectrum is determined
A. Liquid Tissue will be directed toTMSRM/ on the triple quadrupole mass spectrum for the individual chip peptide identified in lysate MRM, which is determined, is applied to the peptide from ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein
I. the optimal retention time of fragment peptide is determined for including but is not limited to the optimum chromatogram condition of following experiment：Gel Electrophoresis, liquid chromatogram, Capillary Electrophoresis, nanometer reverse phase liquid chromatography, high performance liquid chromatography or reverse high performance liquid chromatography
Ii. single isotopic mass of peptide, each propeptide state of charge, each propeptide m/z values, the m/z of each peptide are determined The ionic type of each transition ion of transition ion and each fragment peptide is determined with the SRM/MRM developed for each peptide.
Iii. it may then use that the information from (i) and (ii) carries out SRM/MRM measure on triple quadrupole mass spectrum, wherein Each peptide is respectively provided with the SRM/MRM characteristic peaks of characteristic and uniqueness, and this feature peak is accurately defined on triple quadrupole mass spectrum and carried out Unique SRM/MRM determine
B. carry out SRM/MRM and analyze the function for causing unique SRM/MRM characteristic peak areas from SRM/MRM mass spectral analyses The fragment peptide of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein detected of form contains Amount may indicate that ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein in specified protein lysate Relative amount and absolute content.
Iv. relative quantification can be achieved by the following way：
1. determine ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A by comparing values below The amount increased or decreased of protein：A kind of Liquid Tissue for the biological sample fixed from formalinTMCracking The SRM/MRM characteristic peaks of given ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide detected in thing Area with from least second, third, the biological sample fixed of 4th or more formalin at least second, third, the In four or more Liquid Tissue lysates identical ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The identical SRM/MRM characteristic peak areas of TOPO2A fragment peptides
2. determine ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A by comparing values below The amount increased or decreased of protein：A kind of Liquid Tissue cracking for the biological sample fixed from formalin The SRM/MRM characteristic peaks of given ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide detected in thing Area comes from the SRM/ of the fragment peptide development of the other oroteins in different and individually biological source other samples from origin MRM characteristic peak areas, wherein for the SRM/MRM characteristic peak areas between two kinds of samples of fragments of peptides standard of comparison to each The content for the protein analyzed in sample.
3. determine ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A by comparing values below The amount increased or decreased of protein：Given ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A peptide SRM/MRM characteristic peak areas with the identical Liquid Tissue lysates for the biological sample fixed from formalin Different proteins source other fragment peptides SRM/MRM characteristic peak areas so that by ENT1, ERCC1, FOLR1, RRM1, The change level of TUBB3, TOPO1 and/or TOPO2A protein, which is normalized under the conditions of various kinds of cell, does not change its expression water The level of flat other oroteins.
4. these measure can be applied to ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Unmodified fragment peptide and modification fragment peptide, wherein modification include but is not limited to phosphorylation and/or glycosylation, and wherein with With determining that the relative amount identical mode of unmodified peptide determines the relative level of the peptide of modification.
V. giving the absolute quantitation of peptide can be realized by comparing values below：ENT1 in single biological sample, The SRM/MRM characteristic peak areas of the given fragment peptide of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein The SRM/MRM characteristic peak areas of the interior segments peptide reference material come with incorporation in the protein cleavage thing of biological sample
1. internal standard compound is ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein in measurement Fragment peptide labeled synthesized form.The reference material is mixed in sample with known quantity, and biological sample can be individually determined The SRM/MRM characteristic peak areas of natural fragments of peptides and interior segments peptide reference material in product, then compare two peak areas
2. this can be applied to the fragment peptide of unmodified fragment peptide and modification, wherein these modifications include but is not limited to phosphoric acid Change and/or glycosylate, and the peptide of modification can be wherein determined with determining the abswolute level identical mode of unmodified peptide Abswolute level.
3. by fragment peptide quantitative Application in cancer diagnosis and treatment
A. the fragment peptide level of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein is carried out Relative and/or absolute quantitation and illustrating confirm patient or ENT1 in object tumor tissues, ERCC1, FOLR1, RRM1, The previously determined relevance of TUBB3, TOPO1 and/or TOPO2A protein expression and stage/grade/situation, as in cancer As being fully understood in field
B. the fragment peptide level of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein is carried out Relative and/or absolute quantitation and illustrate relevance from the clinical effectiveness from different therapeutic strategies, wherein the relevance has been Through illustrate in this field or can future explanation (by for patient or groups of objects or the tissue from those patients or object Association Journal of Sex Research).Once the relevance previously established or the relevance drawn in the future are confirmed by the measure, then the measure Method can be used for determining optimal treatment strategy
Internal standard compound can be from just measured ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The labeled synthesized form of the fragment peptide of TOPO2A protein (or includes the labeled synthesis of the fragment peptide discharged after proteolysis The protein or polypeptide of form).The reference material is mixed in sample with known content, and internal sheets in biological sample can be individually determined The SRM/MRM characteristic peak areas of section peptide reference material and native gene peptide, then compare the two peak areas.This can be applied to not repair The fragment peptide of decorations and the fragment peptide of modification, wherein modification includes but is not limited to phosphorylation and/or glycosylation, and can be with determining not The abswolute level identical mode of modified peptides determines the abswolute level of the peptide of modification.
By developing related specific and unique of particular peptide to all peptides are analyzed on triple quadrupole mass spectrum in ion trap Characteristic.The information includes single isotopic mass, its preceding body charge state, precursor m/z values, the transition m/z values of the precursor of the peptide With the ionic type of the transition ion of each identification.The information must be directed in the sample/tissue fixed from formalin Each and each the candidate SRM/MRM peptide directly existed in Liquid Tissue lysates is measured by experiment；Reason It is, it is interesting that simultaneously SRM/MRM of the present invention all can be used in this kind of lysate in peptide of the not all from protein Detection, shows that the peptide not detected can not be considered as exploitation and be used for the Liquid of the quantitative sample/tissue fixed from formalin The candidate peptide that the SRM/MRM of the peptide/protein directly existed in Tissue lysates is determined.
The specific SRM/MRM that particular peptide is carried out on triple quadrupole mass spectrum is determined.Determined and analyzed by specific SRM/MRM Laboratory sample be for example from formalin fix and the tissue of FFPE in the Liquid Tissue protein cleavages that prepare Thing.There is unique SRM/MRM characteristic peaks of the peptide in the sample fixed for formalin in the data display from this kind of measure.
The specific transitions ion characteristic of the peptide is used for specific peptide in the biological sample of quantitative measurment formalin fixation. The absolute content of these data displays peptide, is shown as the function of the molar content of peptide in every microgram institute analysing protein lysate Form.
Based on formalin fix patient source or object origin tissue analysis carry out ENT1, ERCC1, FOLR1, The assessment of RRM1, TUBB3, TOPO1 and/or TOPO2A protein level, which can be provided, is related to diagnosing, in advance for specific patient or object Afterwards to the upper related information for the treatment of.The present invention describe ENT1 in a kind of measurement biological sample, ERCC1, FOLR1, RRM1, The method of TUBB3, TOPO1 and/or TOPO2A protein level, including the use of Mass Spectrometer Method and/or quantitatively by the biological sample In protein digestibility thing prepared by product one or more modifications or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, The content of TOPO1 and/or TOPO2A protein fragments peptides；With calculate ENT1 modified in the sample or unmodified, The level of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein；And wherein described level is relative level Or abswolute level.In related embodiment, quantitative one or more ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 And/or TOPO2A protein fragments peptides is included by relatively being determined compared with the internal standard peptide of the known content of addition in biological sample The content of each ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide, the wherein biological sample Each ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide is with having identical amino in product The internal standard peptide of acid sequence compares.In some embodiments, the internal standard compound is the internal standard peptide of isotope marks, and it includes one kind Or it is a variety of selected from 18O, 17O, 34S,15N、13C、2H or its combination heavy stable isotope.
In measure biological sample of the present invention ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or The method of the level of TOPO2A protein (or alternatively fragment peptide of thing) can be used as the diagnosis of cancer in patient or object Learn indicant.In one embodiment, ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein The measurement result of level can be used for the diagnostic phases/grade/state for determining cancer, and method is discovery in association (such as comparing) tissue ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein level and normal and/or carcinous or cancer The level of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein found in preceding tissue.
The method of specified protein level is to exempt from the Patient Sample A that only detection formalin used is fixed Epidemic disease group (IHC).This method once only analyzes a kind of protein on the single histotomy from specimens sample. Therefore, it is analysis multiple protein quality sample, it is necessary to which, while measuring multiple histotomies, this is time-consuming and laborious.IHC is examined using antibody Survey in the presence of target protein, and any IHC experiments all because of the Potential feasibility that antibody is combined with protein non-specific There is huge intrinsic potential signal background.In addition, IHC is only sxemiquantitative at most.Due to these problems, IHC can not The objective quantification of multiple proteins is provided simultaneously.Current embodiment can use single patient slicing tissue samples with 100% determines the visitor that specificity provides ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein simultaneously See it is quantitative, provided while plenty of time and money is saved it is richer it is valuable be related to ENT1, ERCC1, FOLR1, RRM1, The data of TUBB3, TOPO1 and/or TOPO2A protein expression.
The multiple SRM/MRM determine may additionally include ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or Other additional proteins, including drug target protein matter such as EGFR, IGF-1R and cMet are analyzed beyond TOPO2A protein simultaneously.This It is valuable, because the analysis of additional protein also shows which additional protein can be used for treating specific cancer.Based on volume Erbitux (Erbitux) of the example including targeting EGFR acceptor of the additional protein of outer illustrative drug target protein analysis, Target IGF-1R Figitumumab, and targeting c-Met and VEGF R2 (VEGFR-2) Foretinib。
Because nucleic acid and protein both can be by identical Liquid TissueTMBiomolecule prepared product is analyzed, so The extra letter that can be determined by the nucleic acid generation in the same sample for protein analysis on medical diagnosis on disease and drug therapy Breath.If for example, ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein by some cells to increase Plus horizontal expression, when being determined by SRM, data can provide on cell state and its uncontrollably grow it is potential The information of property, the potential resistance to the action of a drug and cancer development.Meanwhile, can be from identical Liquid TissueTMIn biomolecule prepared product Obtained in the nucleic acid of presence on ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A gene and/or nucleic acid And its information (for example, mRNA molecules and its expression or montage change) of the situation of the protein of coding, can ENT1, It is evaluated while the SRM analyses of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein.Can be To not coming from while the SRM analyses of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A and be present in identical biomolecule prepared product appoint What gene and/or nucleic acid are evaluated.In one embodiment, on ENT1, ERCC1, FOLR1, RRM1, TUBB3, The information of TOPO1 and/or TOPO2A protein and/or one, two, three, four or more of extra protein can lead to Cross and check the nucleic acid of those protein of coding to evaluate.Those nucleic acid can for example by the following method in one or more, two Kind or more is planted or three or more inspections：Sequence measurement, polymerase chain reaction method, pvuii restriction fragment point Analysis, insertion, the identification of missing, and/or with the presence or absence of the determination of mutation, including but not limited to single base is to polymorphism, conversion, top Change or its combination.
The illustrative embodiments of description above and method and composition illustrate the scope of the present invention.However, by It will be apparent to those skilled in the art in change, the invention is not restricted to embodiment described above.
Sequence table
<110>Ai Kepuxun pathological studies company (Expression Pathology Inc)
<120>Determined for the SRM of chemotherapy target
<130> 01152-8034
<160> 103
<170> PatentIn version 3.5
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<213>Homo sapiens (Homo sapiens)
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Asp Ala Gln Ala Ser Ala Ala Pro Ala Ala Pro Leu Pro Glu Arg
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Ile Pro Gln Ser Val Arg
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<213>Homo sapiens
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Ile Leu Gly Ser Leu Val Ala Ile Leu Leu Val Phe Leu Ile Thr Ala
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Ile Leu Val Lys
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Leu Glu Gly Pro Gly Glu Gln Glu Thr Lys
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Leu Asp Leu Ile Ser Lys
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Ser Ser Ile Ala Gly Ser Ser Thr Trp Glu Arg
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Trp Leu Pro Ser Leu Val Leu Ala Arg
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Ser Asn Ser Ile Ile Val Ser Pro Arg
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Gly Asn Pro Val Leu Lys
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Leu Glu Gln Asp Phe Val Ser Arg
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Thr Asp Ser Gln Thr Leu Leu Thr Thr Phe Gly Ser Leu Glu Gln Leu
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Leu Phe Asp Val Leu His Glu Pro Phe Leu Lys
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Ile Ala Trp Ala Arg
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Glu Lys Pro Gly Pro Glu Asp Lys
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Asp Val Ser Tyr Leu Tyr Arg
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Ser Asn Trp His Lys
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His Pro Asp Tyr Ala Ile Leu Ala Ala Arg
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Ile Ala Val Ser Asn Leu His Lys
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Ser Thr Leu Asp Ile Val Leu Ala Asn Lys
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<213>Homo sapiens
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Leu Asn Ser Ala Ile Ile Tyr Asp Arg
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<212> PRT
<213>Homo sapiens
<400> 25
Asp Phe Ser Tyr Asn Tyr Phe Gly Phe Lys
1 5 10
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<212> PRT
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Val Ser Val Gly Ile His Lys
1 5
<210> 27
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<213>Homo sapiens
<400> 27
Glu Asp Ile Asp Ala Ala Ile Glu Thr Tyr Asn Leu Leu Ser Glu Arg
1 5 10 15
<210> 28
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<212> PRT
<213>Homo sapiens
<400> 28
Asp Asp Ser Ile Glu Gly Ile Tyr Asp Thr Leu Lys
1 5 10
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<213>Homo sapiens
<400> 29
Tyr Val Asp Gln Gly Gly Asn Lys
1 5
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<211> 7
<212> PRT
<213>Homo sapiens
<400> 30
Leu Tyr Ala Ser Tyr Glu Lys
1 5
<210> 31
<211> 10
<212> PRT
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Ser Asn Gln Gln Asn Leu Gly Thr Ile Lys
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Leu Ala Glu Val Thr Lys
1 5
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Tyr Pro Phe Glu Ser Ala Glu Ala Gln Leu Leu Asn Lys
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Glu Gln Gly Pro Tyr Glu Thr Tyr Glu Gly Ser Pro Val Ser Lys
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<213>Homo sapiens
<400> 35
Val Leu Ser Gly Glu Phe Gln Ile Val Asn Pro His Leu Leu Lys
1 5 10 15
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<211> 8
<212> PRT
<213>Homo sapiens
<400> 36
Thr Val Trp Glu Ile Ser Gln Lys
1 5
<210> 37
<211> 21
<212> PRT
<213>Homo sapiens
<400> 37
Gly Ala Phe Ile Asp Gln Ser Gln Ser Leu Asn Ile His Ile Ala Glu
1 5 10 15
Pro Asn Tyr Gly Lys
20
<210> 38
<211> 7
<212> PRT
<213>Homo sapiens
<400> 38
His Ser Asn Ser Glu His Lys
1 5
<210> 39
<211> 11
<212> PRT
<213>Homo sapiens
<400> 39
Glu Asn Gly Phe Ser Ser Pro Pro Gln Ile Lys
1 5 10
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<211> 13
<212> PRT
<213>Homo sapiens
<400> 40
Asp Glu Pro Glu Asp Asp Gly Tyr Phe Val Pro Pro Lys
1 5 10
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<211> 8
<212> PRT
<213>Homo sapiens
<400> 41
Leu Glu Glu Glu Glu Asp Gly Lys
1 5
<210> 42
<211> 7
<212> PRT
<213>Homo sapiens
<400> 42
Val Pro Glu Pro Asp Asn Lys
1 5
<210> 43
<211> 6
<212> PRT
<213>Homo sapiens
<400> 43
Trp Trp Glu Glu Glu Arg
1 5
<210> 44
<211> 6
<212> PRT
<213>Homo sapiens
<400> 44
Tyr Pro Glu Gly Ile Lys
1 5
<210> 45
<211> 16
<212> PRT
<213>Homo sapiens
<400> 45
Gly Pro Val Phe Ala Pro Pro Tyr Glu Pro Leu Pro Glu Asn Val Lys
1 5 10 15
<210> 46
<211> 6
<212> PRT
<213>Homo sapiens
<400> 46
Phe Tyr Tyr Asp Gly Lys
1 5
<210> 47
<211> 10
<212> PRT
<213>Homo sapiens
<400> 47
Ala Glu Glu Val Ala Thr Phe Phe Ala Lys
1 5 10
<210> 48
<211> 6
<212> PRT
<213>Homo sapiens
<400> 48
Ala Gln Thr Glu Ala Arg
1 5
<210> 49
<211> 9
<212> PRT
<213>Homo sapiens
<400> 49
Val Pro Ser Pro Pro Pro Gly His Lys
1 5
<210> 50
<211> 16
<212> PRT
<213>Homo sapiens
<400> 50
Val Thr Trp Leu Val Ser Trp Thr Glu Asn Ile Gln Gly Ser Ile Lys
1 5 10 15
<210> 51
<211> 24
<212> PRT
<213>Homo sapiens
<400> 51
Val Glu His Ile Asn Leu His Pro Glu Leu Asp Gly Gln Glu Tyr Val
1 5 10 15
Val Glu Phe Asp Phe Leu Gly Lys
20
<210> 52
<211> 9
<212> PRT
<213>Homo sapiens
<400> 52
Gln Pro Glu Asp Asp Leu Phe Asp Arg
1 5
<210> 53
<211> 8
<212> PRT
<213>Homo sapiens
<400> 53
Leu Asn Thr Gly Ile Leu Asn Lys
1 5
<210> 54
<211> 12
<212> PRT
<213>Homo sapiens
<400> 54
Glu Leu Thr Ala Pro Asp Glu Asn Ile Pro Ala Lys
1 5 10
<210> 55
<211> 7
<212> PRT
<213>Homo sapiens
<400> 55
Glu Gln Leu Ala Asp Ala Arg
1 5
<210> 56
<211> 8
<212> PRT
<213>Homo sapiens
<400> 56
Leu Glu Val Gln Ala Thr Asp Arg
1 5
<210> 57
<211> 8
<212> PRT
<213>Homo sapiens
<400> 57
Gln Ile Ala Leu Gly Thr Ser Lys
1 5
<210> 58
<211> 7
<212> PRT
<213>Homo sapiens
<400> 58
Trp Gly Val Pro Ile Glu Lys
1 5
<210> 59
<211> 11
<212> PRT
<213>Homo sapiens
<400> 59
Ser Gln Ser Ser Thr Ser Thr Thr Gly Ala Lys
1 5 10
<210> 60
<211> 12
<212> PRT
<213>Homo sapiens
<400> 60
Ser Ser Asp Glu Ser Asn Phe Asp Val Pro Pro Arg
1 5 10
<210> 61
<211> 8
<212> PRT
<213>Homo sapiens
<400> 61
Gly Tyr Asp Ser Asp Pro Val Lys
1 5
<210> 62
<211> 14
<212> PRT
<213>Homo sapiens
<400> 62
Val Pro Asp Glu Glu Glu Asn Glu Glu Ser Asp Asn Glu Lys
1 5 10
<210> 63
<211> 8
<212> PRT
<213>Homo sapiens
<400> 63
Glu Gln Glu Leu Asp Thr Leu Lys
1 5
<210> 64
<211> 7
<212> PRT
<213>Homo sapiens
<400> 64
Ser Pro Ser Asp Leu Trp Lys
1 5
<210> 65
<211> 16
<212> PRT
<213>Homo sapiens
<400> 65
Glu Asp Leu Ala Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys
1 5 10 15
<210> 66
<211> 10
<212> PRT
<213>Homo sapiens
<400> 66
Gln Asp Glu Gln Val Gly Leu Pro Gly Lys
1 5 10
<210> 67
<211> 9
<212> PRT
<213>Homo sapiens
<400> 67
Val Ile His Glu Gln Val Asn His Arg
1 5
<210> 68
<211> 15
<212> PRT
<213>Homo sapiens
<400> 68
Gly Phe Gln Gln Ile Ser Phe Val Asn Ser Ile Ala Thr Ser Lys
1 5 10 15
<210> 69
<211> 12
<212> PRT
<213>Homo sapiens
<400> 69
His Val Asp Tyr Val Ala Asp Gln Ile Val Thr Lys
1 5 10
<210> 70
<211> 6
<212> PRT
<213>Homo sapiens
<400> 70
Leu Val Asp Val Val Lys
1 5
<210> 71
<211> 6
<212> PRT
<213>Homo sapiens
<400> 71
Gly Gly Val Ala Val Lys
1 5
<210> 72
<211> 7
<212> PRT
<213>Homo sapiens
<400> 72
Ala Gln Val Gln Leu Asn Lys
1 5
<210> 73
<211> 10
<212> PRT
<213>Homo sapiens
<400> 73
Leu Asp Asp Ala Asn Asp Ala Gly Gly Arg
1 5 10
<210> 74
<211> 12
<212> PRT
<213>Homo sapiens
<400> 74
Thr Leu Ala Val Ser Gly Leu Gly Val Val Gly Arg
1 5 10
<210> 75
<211> 7
<212> PRT
<213>Homo sapiens
<400> 75
Tyr Gly Val Phe Pro Leu Arg
1 5
<210> 76
<211> 7
<212> PRT
<213>Homo sapiens
<400> 76
Ile Val Gly Leu Gln Tyr Lys
1 5
<210> 77
<211> 9
<212> PRT
<213>Homo sapiens
<400> 77
Asn Tyr Glu Asp Glu Asp Ser Leu Lys
1 5
<210> 78
<211> 16
<212> PRT
<213>Homo sapiens
<400> 78
Gly Leu Leu Ile Asn Phe Ile His His Asn Trp Pro Ser Leu Leu Arg
1 5 10 15
<210> 79
<211> 11
<212> PRT
<213>Homo sapiens
<400> 79
Phe Leu Glu Glu Phe Ile Thr Pro Ile Val Lys
1 5 10
<210> 80
<211> 7
<212> PRT
<213>Homo sapiens
<400> 80
Ser Ser Thr Pro Asn His Lys
1 5
<210> 81
<211> 8
<212> PRT
<213>Homo sapiens
<400> 81
Gly Leu Gly Thr Ser Thr Ser Lys
1 5
<210> 82
<211> 16
<212> PRT
<213>Homo sapiens
<400> 82
Tyr Ser Gly Pro Glu Asp Asp Ala Ala Ile Ser Leu Ala Phe Ser Lys
1 5 10 15
<210> 83
<211> 25
<212> PRT
<213>Homo sapiens
<400> 83
Leu Leu Gly Leu Pro Glu Asp Tyr Leu Tyr Gly Gln Thr Thr Thr Tyr
1 5 10 15
Leu Thr Tyr Asn Asp Phe Ile Asn Lys
20 25
<210> 84
<211> 12
<212> PRT
<213>Homo sapiens
<400> 84
Glu Leu Ile Leu Phe Ser Asn Ser Asp Asn Glu Arg
1 5 10
<210> 85
<211> 8
<212> PRT
<213>Homo sapiens
<400> 85
Phe Leu Tyr Asp Asp Asn Gln Arg
1 5
<210> 86
<211> 7
<212> PRT
<213>Homo sapiens
<400> 86
Ile Pro Asn Phe Asp Val Arg
1 5
<210> 87
<211> 7
<212> PRT
<213>Homo sapiens
<400> 87
Glu Ile Val Asn Asn Ile Arg
1 5
<210> 88
<211> 7
<212> PRT
<213>Homo sapiens
<400> 88
Thr Trp Thr Gln Thr Tyr Lys
1 5
<210> 89
<211> 9
<212> PRT
<213>Homo sapiens
<400> 89
Thr Pro Pro Leu Ile Thr Asp Tyr Arg
1 5
<210> 90
<211> 9
<212> PRT
<213>Homo sapiens
<400> 90
Glu Tyr His Thr Asp Thr Thr Val Lys
1 5
<210> 91
<211> 5
<212> PRT
<213>Homo sapiens
<400> 91
Val Gly Leu His Lys
1 5
<210> 92
<211> 9
<212> PRT
<213>Homo sapiens
<400> 92
Tyr Asp Thr Val Leu Asp Ile Leu Arg
1 5
<210> 93
<211> 6
<212> PRT
<213>Homo sapiens
<400> 93
Asp Phe Phe Glu Leu Arg
1 5
<210> 94
<211> 10
<212> PRT
<213>Homo sapiens
<400> 94
Glu Val Thr Phe Val Pro Gly Leu Tyr Lys
1 5 10
<210> 95
<211> 13
<212> PRT
<213>Homo sapiens
<400> 95
Ile Phe Asp Glu Ile Leu Val Asn Ala Ala Asp Asn Lys
1 5 10
<210> 96
<211> 17
<212> PRT
<213>Homo sapiens
<400> 96
Val Thr Ile Asp Pro Glu Asn Asn Leu Ile Ser Ile Trp Asn Asn Gly
1 5 10 15
Lys
<210> 97
<211> 8
<212> PRT
<213>Homo sapiens
<400> 97
Gly Ile Pro Val Val Glu His Lys
1 5
<210> 98
<211> 6
<212> PRT
<213>Homo sapiens
<400> 98
Asn Gly Tyr Gly Ala Lys
1 5
<210> 99
<211> 8
<212> PRT
<213>Homo sapiens
<400> 99
Phe Thr Val Glu Thr Ala Ser Arg
1 5
<210> 100
<211> 9
<212> PRT
<213>Homo sapiens
<400> 100
Ala Tyr Asp Ile Ala Gly Ser Thr Lys
1 5
<210> 101
<211> 7
<212> PRT
<213>Homo sapiens
<400> 101
Val Phe Leu Asn Gly Asn Lys
1 5
<210> 102
<211> 17
<212> PRT
<213>Homo sapiens
<400> 102
Met Ser Ser Thr Phe Ile Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
1 5 10 15
Lys
<210> 103
<211> 25
<212> PRT
<213>Homo sapiens
<400> 103
Leu Ala Thr Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Ala Thr
1 5 10 15
Met Ser Gly Val Thr Thr Ser Leu Arg
20 25

Claims (17)

1. ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein water in one kind measurement biological sample Flat method, including the use of one or more in Mass Spectrometer Method and/or the quantitative protein digestibility thing from the biological sample The content of modification and/or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A fragment peptide； With ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A modified in the calculating sample or unmodified The level of protein；And
Wherein described content is relative amount or absolute content.
2. the method described in claim 1, it further comprises being modified in detection and/or quantitative one or more or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide before the protein is disappeared The step of compound is classified.
3. the method described in claim 1, wherein the protein digestibility thing of the biological sample passes through Liquid TissueTMIt is prepared by scheme.
4. the method described in claim 1, wherein the protein digestibility thing includes protease digestion thing.
5. the method described in claim 1, wherein the mass spectrum include tandem mass spectrum, ion trap mass spectrometry, triple quadrupole mass spectrum, MALDI-TOF mass spectrums, MALDI mass spectrums and/or flight time mass spectrum.
6. the method any one of claim 1-5, wherein described ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 And/or TOPO2A protein fragments peptide includes the amino acid sequence shown in following sequence：SEQ ID NO：1、SEQ ID NO：2、 SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48、SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56、SEQ ID NO：57、SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64、SEQ ID NO：65、SEQ ID NO：66、SEQ ID NO：67、SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74、SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78、SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98、SEQ ID NO：99、SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102 and SEQ ID NO：103.
7. the method any one of claim 1-5, wherein the biological sample is blood sample, urine samples, serum sample Product, ascites sample, tired liquid sample, lymph, saliva sample, cell or solid tissue.
8. the method any one of claim 1-5, its further comprise quantitative modification and/or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide.
9. the method described in claim 8, wherein quantitative described ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide include one or more ENT1 in a kind of relatively biological sample, ERCC1, FOLR1, RRM1, The content of TUBB3, TOPO1 and/or TOPO2A protein fragments peptide from it is different and individually identical ENT1 in biological sample, The content of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide, the protein fragments peptide bag About 8 to about 45 amino acid residues containing ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein Amino acid sequence, and it is shown in SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、 SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48、SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56、SEQ ID NO：57、SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64、SEQ ID NO：65、SEQ ID NO：66、SEQ ID NO：67、SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74、SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78、SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98、SEQ ID NO：99、SEQ ID NO：100、SEQ ID NO：101、 SEQ ID NO：102 and SEQ ID NO：103.
10. the method described in claim 9, wherein quantitative one or more ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptides is included by relatively determining biology compared with the internal standard peptide of the known content of addition The content of each ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide in sample, wherein, Each ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide in the biological sample with Internal standard peptide with same amino acid sequence compares.
11. the method described in claim 10, wherein the internal standard peptide is the peptide of isotope marks.
12. the method any one of claim 1-5, wherein a kind of in detection and/or the quantitative protein digestibility thing A variety of modifications or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments The content of peptide, which is shown, has modifying or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 in patient or object And/or TOPO2A protein and associated with cancer including lung cancer.
13. the method described in claim 12, it further comprises associating the detection and/or quantifies one or more modifications Or content or the institute of unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide The result of content of ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein is stated with including lung cancer Diagnostic phases/grade/state of cancer.
14. the method described in claim 13, wherein associating the detection and/or quantitative one or more modifying or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide content or the ENT1, The result of the content of ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein and the diagnostic phases of cancer/etc. The content of peptide of the level/state with detection and/or other quantitative protein or from other protein is combined with multiple form, so that The extraneous information of diagnostic phases/grade/state on the cancer including lung cancer is provided.
15. the method described in claim 13, in addition to give to patient or object the therapeutic agent of therapeutically effective amount, the biology Sample is obtained from the patient or object, wherein the therapeutic agent and/or the content of the therapeutic agent given are based on one kind or many Plant modification or unmodified ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein fragments peptide The content of content or ENT1, ERCC1, FOLR1, RRM1, TUBB3, TOPO1 and/or TOPO2A protein.
16. the method described in claim 15, wherein the treatment or the therapeutic agent for expression ENT1, ERCC1, FOLR1, The cancer cell of RRM1, TUBB3, TOPO1 and/or TOPO2A protein.
17. a kind of composition, its comprising it is one or more, two or more, it is three or more, four kinds or more plant, five Kind or more plant, six kinds or more plant, seven kinds or more plant, eight kinds or more plant, nine kinds or more kinds, or ten kinds or more plant Peptide (the SEQ ID NO of table 1：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO： 12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28、SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48、SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56.SEQ ID NO：57、SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60、SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64、SEQ ID NO：65、SEQ ID NO：66、SEQ ID NO：67、SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70、SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74、SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78、SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98、SEQ ID NO：99、SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102 and SEQ ID NO：And/or its antibody 103).
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